Immunoassay device and method

ABSTRACT

A device and related method for performing one or more immunoassays to detect the presence of respective analytes in a sample. The device involves the use of a unitary bibulous material providing one or more flow paths having a common origin site and a plurality of respective reagent zones providing the reagents necessary for performing a visual readout, competitive immunoassay for the presence of the respective analyte.

TECHNICAL FIELD

The present invention relates to the field of diagnostic immunoassaysand related devices for carrying out such assays. In another aspect, theinvention relates to assays for analytes such as drugs of abuse or theirmetabolites. In another aspect, the invention relates to devices ormeans for simultaneously carrying out multiple assays for differentanalytes within a single sample.

BACKGROUND OF THE INVENTION

The use of analytical assays, including those used to determine thepresence of drugs of abuse, has grown rapidly over the past decade. By1993, the U.S. drug-testing market, alone, was estimated to be at least$500M. The drug-testing industry is poised for further growth as aresult of new federal U.S. regulations that will significantly increasethe number of workers subject to testing for drug and alcohol abuse.

At present, most drug testing involves sample collection followed byinstrument-based "wet chemistry" laboratory analysis. However, theon-site, or "point of care" market has been growing rapidly over thepast two years. Although no current figures are available, the marketfor non-instrumented immunoassay-based drugs of abuse test kits appearsto be growing at the rate of 20-40% per year.

Currently, there are a number of single analyte immunoassay-based drugsof abuse diagnostic tests on the market. These include tests produced byRoche Diagnostic Systems, Hansen Hong Biomedical Co. Ltd., DrugScreening Systems, Editek, Inc., Hycor Biomedical, U.S. Drug TestingInc., Thermedics Detection, Inc., and Fingerprint Biotek. Such devicesgenerally work well for situations in which a specific drug issuspected. In many cases, however, such as in emergency room settings,it would be particularly desireable to have a rapid, self-performingassay for one or more drugs that may be present in a given patient.

A variety of assay kits have been described having the capability toperform diagnostic assays. For instance, a series of patents issued toOlson (U.S. Pat. Nos. 4,959,307; 4,963,468; 5,085,987; and 5,085,988)relate to an immunoseparating strip having a bibulous material, anondiffusively bound first receptor, and a nondiffusively bound secondreceptor. In each embodiment, however, the method of using the devicerequires the first step of preparing a separate test solution containingthe sample, antibody for the analyte, and a conjugate of analyte and alabel. Once formed, the competitive reaction progresses to the desiredextent in the solution phase. The solution is then transferred by theuser to the contact portion of the analytical device, where it beginsits flow along the path. (See, e.g., the U.S. Pat. No. '987, col. 13,lines 35-38 and col 20, lines 10-11.)

Others have disclosed the use of kits capable of performing two or moreassays, including multi-analyte on-site formats. A kit available fromBiosite ("Triage" brand), is said to allow the differential detection ofthe presence of several common drugs of abuse in a single urine or serumsample. See, for example, Buechler, et al, Clin. Chem. 38(9):1678-1684(1992). At least one drawback of this device is the need to separatelyadd sample to a region containing lyophilized reagents, where it is leftfor a period of time (e.g, 10 minutes), in order to allow the sample toreconstitute and equilibrate with the reagents.

This and other single and multi-analyte test kits currently on themarket have several drawbacks. Present formats tend to be quite complex,with specific affinity constants playing a key role in the competitivebinding reactions. Moreover, the formats can suffer from false resultsif the patient is on high doses of the analyte drug. Also, presentformats typically require exact reagent concentrations (i.e., ratios ofanalyte to anti-analyte), which can be compromised if one member of theligand-receptor pair begins to deteriorate.

Particularly troublesome are kits that rely on the use of an immobilizedantibody or binding reagent, where the amount of this reagent needs tobe rigidly controlled. The binding capacity of immobilized receptors canbe highly dependent on the particular immobilization methods andconditions. This dependence causes the manufacture of such assays to beunpredictable and difficult to reproduce. Shelf life stability can beaffected as well.

What is clearly needed is an analyte test kit that can be used for anumber of different drugs of abuse, and particularly one that can besimultaneously used to perform a plurality of assays, in a manner thatis easier to manufacture and simple and reliable in use.

BRIEF DESCRIPTION OF THE DRAWING

In the Drawing:

FIG. 1 depicts a diagram of a preferred embodiment of the presentinvention, having a single analyte, immunoassay-based on-site testformat.

FIG. 2 depicts a diagram of a preferred embodiment of the presentinvention, having a multiple analyte, immunoassay-based on-site testformat.

SUMMARY OF THE INVENTION

The present invention provides a device and related method forperforming one or more competitive immunoassays to detect the presenceof respective analytes in a sample. The device can be provided as aunitary device, i.e., in which each of the steps and reagents areself-contained on the device. Preferably, each of the steps areself-performing, that is they proceed autonomously upon the applicationof a sample. In a particularly preferred embodiment, once the sample hasbeen applied to the device, the entire assay (including competitivereaction), progress solely by steps that involve capillary flow fromeach site or zone to the next. As a result, such a device can be usedwithout the need to separately perform a competitive assay reaction in asolution, or physically transfer the solution in a non-capillary mannerto a separate device or zone.

In a preferred embodiment, the device comprises a bibulous materialproviding one or more flow paths, each flow path comprising:

(a) an origin site for the application of a fluid sample,

(b) a plurality of reagent zones emanating from the origin site andproviding the reagents necessary for performing a visual readout,competitive immunoassay for the presence of the respective analyte, thezones comprising, in order and in the direction of flow:

(i) a competition zone in fluid communication with the origin site andcomprising a detectable analyte-conjugate and binding partner for theanalyte, both being diffusively positioned in such a manner that freeanalyte present in the sample is capable of binding to the bindingpartner in a competitive manner with the analyte-conjugate;

(ii) a retention zone in latent fluid communication with the competitionzone and comprising an excess amount of a nondiffusively bound capturereagent capable of binding to free or bound binding partner in order toremove it from continued flow in the flow path; and

(iii) a readout zone in fluid communication with the retention zone andcomprising nondiffusively bound receptor capable of binding toanalyte-conjugate, but not to unconjugated analyte, in a detectablemanner.

In the course of using a device as described above, the presence ofincreasing amounts of free analyte in a sample corresponds withdecreased binding of analyte-conjugate to the binding partner. In turn,an increased amount of free analyte-conjugate is able to continue downthe flow path. Ultimately, the free analyte-conjugate becomesnondiffusively bound to a corresponding binding partner in the readoutzone, and is there detected in order to provide a positive indication ofthe presence of analyte in the original sample.

In a preferred embodiment the device and method can be used tosimultaneously perform a plurality of immunoassays to detect thepresence of respective analytes in a sample. In such a preferred"multi-analyte" embodiment, the device comprises a bibulous materialproviding one or more flow paths, each flow path comprising:

(a) a common origin site on the bibulous material for the simultaneousapplication of a fluid sample,

(b) a plurality of respective reagent zones in active or latent fluidcommunication with the origin, the reagent zones of each flow pathproviding the reagents necessary for performing a visual readout,competitive immunoassay for the presence of the respective analyte, thezones for each flow path comprising, in order and in the direction offlow:

(i) a competition zone in fluid communication with the origin andcomprising a detectable analyte-conjugate and binding partner for therespective analyte, both the conjugate and binding partner beingdiffusively positioned in such a manner that any free analyte present inthe sample is capable of binding to the binding partner in a competitivemanner with the analyte-conjugate;

(ii) a retention zone in latent fluid communication with a respectivecompetition zone and comprising an excess amount of a nondiffusivelybound capture reagent capable of binding to free or bound bindingpartner in order to remove it from continued flow in the flow path; and

(iii) a readout zone in fluid communication with a respective retentionzone, and comprising nondiffusively bound receptor capable of binding toanalyte-conjugate, but not to unconjugated analyte, in a detectablemanner.

The flow paths preferably further each comprise one or more positiveand/or negative procedural control zones and reagents. In one preferredembodiment, the device further comprises a terminal site downstream fromthe sequential reagent zones and comprising an indicator reagent forconfirming the completion of the respective assay.

In a particularly preferred embodiment, the device comprises an inertholder comprising top and bottom portions for supporting the bibulousmaterial, the top portion providing an opening for fluid sample accessto the common origin, as well as openings for viewing the respectivereadout and terminal indicator sites along each flow path.

In one embodiment, the flow paths for a plurality of assays can beprovided in an overlapping and/or side-by-side manner, and in the samedirection along the bibulous material. In an alternative embodiment, thedevice provides a separate, discrete flow path for each analyte, theflow paths being positioned to extend in a radial direction from thecommon origin.

In a particularly preferred embodiment, the present invention provides amulti-analyte assay capable of differentially detecting the presence ofone to four drug(s) in a single sample of urine or serum. Such analytesinclude tetrahydrocannabinol (THC), cocaine, opiates, and amphetamines,which are detected without interference from the other analytes.

DETAILED DESCRIPTION

In the present specification, the following words and phrases will havethe meaning ascribed to them:

"zone" will refer to a discrete situs containing one or more reagentsand positioned along the flow path of a particular assay, each zone orsitus having a surface area less than that of the bibulous material;

"downstream", as applied to zones, will refer to a zone that is flowablyseparated from the preceding zone, and in the direction of flow of asample. In a typical embodiment, for instance, each zone will be on theorder of one or more millimeters from each other. Additionally, theremay be two or more discrete regions within zones, such as the regionscarrying analyte-conjugate and binding partner, respectively in thecompetition zone. Alternatively, with non-interfering reagents, zonesand/or regions within zones may occasionally be positioned in anoverlapping configuration with preceding and/or following zones.

"unitary", as applied to the bibulous material, means that a singleaqueous sample can be added to the origin and progress by active orlatent capillary flow through each of the zones along a respective flowpath. "Bibulous", in turn, refers to a material, or combination ofmaterials, sufficient to permit the capillary flow of sample from theorigin and through each reagent zone.

"simultaneous" will mean that each of a plurality of assays on amulti-analyte device are capable of being performed at substantially thesame time and by the application of a single sample to the origin.

"competitive" will mean that the amount of analyte-conjugate thatbecomes bound to free binding partner is dependent upon and related tothe presence or absence of analyte in the sample.

"nondiffusively bound" will be used interchangeably with the word"immobilized" to describe reagents that are stably retained in aparticular zone under conditions of use.

The present invention provides a device and method for simultaneouslyperforming one or more immunoassays to detect the presence of respectiveanalytes in a sample. Suitable analytes include those capable of beingprovided in the form of a conjugate. Alternatively, the analyte can bein the form of a derivative or metabolite of the compound of interest.

Generally, an analyte is any compound to be detected that is capable ofbeing bound by a receptor, and capable of being recovered in syntheticand/or purified form sufficient to allow it to be conjugated and used ina competitive assay with sample analyte and the receptor. Thesecompounds include mono-epitopic analytes of relatively small molecularweight (e.g., about 100 to 2000), and poly-epitopic antigens of largermolecular weight (e.g., greater than about 2000). Representativeanalytes are those described, for instance, in U.S. Pat. Nos. 4,299,916and 4,275,149, the disclosures of both of which are incorporated hereinby reference.

Examples of suitable analytes include, but are not limited to,pesticides and their metabolites and derivatives (e.g., polyhalogenatedbiphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenatedsulfonamides), and drugs and their metabolites and derivatives (e.g.,alkaloids, steroids, lactams, aminoalkylbenzenes, benzheterocyclics,purines, vitamins, antibiotics, nucleosides and nucleotides, drugsderived from marijuana, and miscellaneous drugs).

The device and method of the present invention can be used with anysuitable sample. Generally, and preferably, the sample is an aqueous onethat is obtained directly from the source (e.g., urine or blood).Alternatively, the sample can be prepared by mixing or extracting anon-aqueous sample (e.g., tissue) with an aqueous solvent (e.g.,buffered solution). Generally, the sample is any substance suspected ofcontaining the compound or compounds of interest. This includes theanalysis of ground water for contaminants, the analysis of agriculturalproducts for naturally occurring toxic agents such as aflatoxin, and thelike.

Occasionally, analyses of this type will require an extraction step inwhich a sample is mixed with a liquid extraction media which can beaqueous, organic, or an aqueous/organic mixture. Upon extraction of thematerial of interest, the extracting solution itself can be used as thesample and can be evaluated directly or concentrated, diluted,evaporated, and reconstituted, etc. before evaluation in the instantdevice.

Additional examples of evaluations requiring extraction includepesticide residues, bacterial metabolites or other contaminants in meator seafood, and herbicide residues or other pollutants in soil samples.

A preferred device of this invention comprises a unitary bibulousmaterial providing one or more flow paths. Preferably, although notnecessarily, the bibulous material will actually be provided in the formof a single, integral material. Alternatively, the bibulous can formedby overlapping or abutting otherwise discrete materials. Examples ofsuitable bibulous materials include (nitrocellulose membranes, nylonmembranes, or other commercially available membranes).

Bibulous materials useful in the instant device include porous materialsthat are susceptible to being traversed by an aqueous medium in responseto capillary force. Such materials are generally hydrophilic or arecapable of being rendered hydrophilic and include inorganic powders,such as silica and alumina; natural polymeric materials particularlycellulosic materials such as filter paper, chromatographic paper and thelike; synthetic or modified naturally occurring polymers such asnitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide,crosslinked dextran, agarose, etc.; either used alone or in conjunctionwith other materials. A preferred bibulous material includes glass fiberfilter paper. The bibulous material can be attached to a support, or mayprovide its own support. The bibulous material may contain functionalgroups, or be capable of being functionalized to permit covalent bondingor receptors to other moieties.

When two or more flow paths are employed in a single device, preferablyeach path comprises a common origin site on the bibulous material forthe simultaneous application of a fluid sample. As can be seen, theorigin is "common" in that the application of a single sample serves tobegin the flow of sample simultaneously in each flow path.

Each flow path in a device of the present invention further comprises aplurality of respective reagent zones at or downstream from the originsite. The reagent zones of each immunoassay provide the reagentsnecessary for performing a visual readout, competitive immunoassay forthe presence of the respective analyte. In a preferred embodiment, thereagent zones provide each of the reagents necessary, i.e., withouthaving to physically remove and reapply reagents (e.g., using a pipet)along the path or in the course of the assay.

The zones of a particular flow path comprise, in the order and directionof flow, a competition zone comprising analyte-conjugate and bindingpartner for the analyte, both being diffusively positioned in such amanner that any free analyte present in the sample is capable of bindingto the binding partner in a competitive manner with the analyteconjugate.

The competition zone can itself serve as the origin site for theapplication of sample, or it can be downstream from the origin itself.Additionally, the various reagents present in the competition zone canbe staggered or arranged in any suitable manner, in order to alter oraffect the kinetics of the competition reaction.

For instance, the binding partner can be positioned upstream of theanalyte conjugate, in order to allow the analyte to react with thebinding partner before the binding partner is exposed to the analyteconjugate. This approach will provide the analyte with a competitiveadvantage over the conjugate for binding sites on the binding partner.In turn, this approach can be used to increase the sensitivity ofdetection for a particular analyte.

The binding partner is preferably a receptor for the analyte. A receptoris any compound or composition capable of recognizing a particularspatial and polar organization of the analyte of interest. Illustrativereceptors include naturally occurring receptors; e.g., antibodies,enzymes, lectins, and the like. A preferred receptor for the analyte isan antibody to the analyte. An antibody is an immunoglobulin orderivative or fragment thereof having an area on the surface or in acavity which specifically binds to and is thereby defined ascomplimentary with a particular spatial and polar organization ofanother molecule. The antibody can be monoclonal or polyclonal, and canbe prepared by techniques that are well known in the art, such asimmunization of a host and collection or sera or hybrid cell linetechnology.

Alternatively, the binding reagent can itself take the form of a complexof molecules, for instance a complex of a first antibody for the analyteand second antibody specific for the first antibody. The components ofsuch a complex can be separately provided either in the competition zoneand/or at any suitable location leading to the retention zone. Suchcomplexes can be useful, for instance, in improving the removal ofbinding reagent in the retention zone by increasing its size or affinityfor the capture reagent.

The analyte-conjugate is generally provided in the form of a label ortracer, for example a catalyst, usually an enzyme, conjugated to theanalyte. A label can be any molecule or system of molecules bound orconjugated to the analyte that is capable of producing a perceptiblesignal. A preferred label consists of colloidal metal particles, or solparticles that are colored. Those skilled in the art will recognize thatmany elements are capable of functioning as a label, including, withoutlimitation, radionuclides, fluorescent species, phosphorescent species,chemiluminescent materials, dyes, enzymes, sol particles, coloredpolymeric materials, and the like. Based upon the known binding kineticsof the monoclonal anti-drug antibodies, standard techniques can be usedto prepare drug conjugates having the correct epitope available forantibody binding.

A flow path further comprises a retention zone in latent fluidcommunication with the competition zone. By "latent fluid communication"it is meant that the fluid communication between the competition zoneand retention zone can be sufficiently delayed, and/or the reaction ratesufficiently increased, in order to allow the competition reaction toprogress to a desired extent. In addition to increasing the reactiontime within the competition zone, the extent of reaction can be improvedby other means as well, such as by elevating the temperature of thebinding reaction and/or by improving the mixing characteristics of thereagents (e.g., by effervescence).

The delay means for achieving latent capillary flow can be of a passivenature (i.e., occurring automatically in the course of use), or of anactive nature (e.g., requiring some act on the part of the user). Giventhe present description, those skilled in the art will appreciate themanner in which delay means can be provided and used in a device of thepresent invention. See, for instance, PCT application Nos. WO 92/21434and WO 93/24231, the disclosures of which are incorporated herein byreference.

In a suitable approach, the residence time of the solution in thecompetition zone is increased by configuring the respective competitionand retention zones (and/or an interface between them) in such a mannerthat the flow of solution from the competition zone is controllablydelayed. Examples of suitable delay means include, but are not limitedto, the use of a dissolvable barrier, a permeable physical barrier, oran expandable bridging material.

For example, a porous or dissolvable barrier can be interspersed betweenthe zones in order to allow the solution to reside in the competitionzone for a sufficient period of time before the barrier is itselfpermeated or dissolved. Similarly, the capillary flow path itself can bemade to take a tortuous route between the zones, thereby increasing theeffective reaction time for the assay. As yet another example, ahydratable, expandable (e.g., sponge-like) material can be employed inthe competition zone, in such a manner that the material is initiallynot in physical contact with the retention zone. Upon hydration with thetest solution, and in the course of allowing the competitive reaction tooccur, the material can expand or swell to a point where it makesphysical contact and permits fluid communication with the retentionzone.

In an alternative embodiment, the latent capillary flow between thecompetition and retention zones can be actively initiated by the user,e.g., by manipulating or releasing a barrier between zones, forming acapillary bridge between them, or physically placing (e.g., pinching)zones together. See, for instance, U.S. Pat. No. 4,826,759, thedisclosure of which is incorporated herein by reference.

The retention zone comprises an excess amount of a nondiffusively boundcapture reagent capable of binding to free or bound binding partner inorder to remove it from continued flow in the flow path. This capturereagent is a typically a receptor capable of binding to the bindingpartner. A preferred capture reagent is an antibody capable of bindingto the binding partner.

Since the binding partner is preferably an antibody to the analyte, thecapture reagent is preferentially an antibody capable of binding to theantibody for the analyte. It can be, for example, an antibody raised ina different species than that used to raise the antibody for theanalyte. The capture reagent can also be a receptor, such as protein A,which binds to a particular site on the immunoglobulin molecule. Inanother embodiment, the binding partner can be coupled to a molecule,such as biotin, and the capture reagent can be specific for such amolecule, for example, antibiotin or avidin.

The approach of the present invention provides a particular advantageover many conventional assays employing immobilized receptors. Asdescribed above, the binding capacity of such immobilized receptors canbe dependent upon the immobilization methods and conditions, such thatthe manufacture of such assays is unpredictable and difficult toreproduce. In the present invention, the immobilized capture reagent canbe present in an excess amount, thus permitting the development of anassay with predictable performance characteristics.

The immobilization of capture reagent is sufficiently stable, in orderto prevent the dissociation of reagent from the material, which in turncould lead to false results. As a further safeguard against error causedby disassociated capture reagent, those skilled in the art willappreciate the manner in which an additional reagent can be employed inorder to itself bind and retain disassociated capture reagent. Onesuitable approach involves the use of a second retention zone, e.g, ator between the first retention zone and the readout zone, havingimmobilized second capture reagent specific for the first capturereagent (i.e., the capture reagent provided in the retention zone).

Lastly, a flow path further comprises a readout zone comprisingnondiffusively bound receptor capable of binding to analyte-conjugatebut not to unconjugated analyte in a detectable manner.

The nondiffusively bound receptor is capable of binding to theanalyte-conjugate, but not to the analyte alone. This receptor bindsthen, either to the label portion of the conjugate, or to an additionalmoiety provided by the conjugate (e.g., by virtue of the binding ofanalyte and label) but absent from the analyte alone. A preferrednondiffusively bound receptor is an antibody capable of binding to thespacer moiety that is used to conjugate the analyte to the label.

In a particularly preferred embodiment, the readout is provided in theform of the completion of a plus ("+") sign indicating the presence ofanalyte. In such an embodiment, the minus ("-") portion of the readoutcan be provided by any suitable means.

In one embodiment, the minus portion is provided by the use ofadditional reagents positioned along the flow path. For instance, adetectable conjugate is positioned along the flow path, and preferablyat the origin itself. The minus portion of the readout can be providedin the form of a nondiffusively bound antibody to the detectableconjugate. For example, when a conjugate of gold-KLH is present indiffusive form, with a nondiffusively bound anti-KLH antibody formingthe minus portion of the readout zone.

Lastly, the flow path preferably further comprises a terminal sitedownstream from the sequential reagent zones and comprising an indicatorreagent for confirming the completion of the respective assay. Theindicator reagent is typically a material that is sensitive to thepresence of the sample. It is generally a material that will changecolor in response to the presence of some moiety in the sample solution.Examples of such a reagent include pH indicator dyes, dyes sensitive tothe presence of proteins, and dyes sensitive to hydration states.

The device of the present invention can be of any suitable form anddimensions in order to achieve the desired purpose. In a preferredembodiment, the device is provided in the form of an inert holdercomprising top and bottom portions for supporting the bibulous material,the top portion providing an opening for fluid sample access to thecommon origin, as well as openings for viewing the respective readoutand terminal indicator sites along each flow path.

The manufacture of a typical design of the present test format will bedescribed with reference to the Drawing, including FIG. 1 (singleanalyte format) and FIG. 2 (multianalyte format). To the user, the testof FIG. 2 would appear as a single test device 10 having paths 12 thatemanate and readout in each of four directions. Paths 12 each comprise acompetition zone 22 (as shown, having discrete regions therein foranalyte-conjugate and first binding partner, respectively), retentionzone 24 and readout zone 26. The sample is added to the center well 14,and the test would be complete when the four completion indicatorwindows 16 change color. If the assay is operating properly, thenegative portion 18 of the sign appears as a color change for each ofthe analytes. If the patient is drug positive for any of the analytes,then the respective plus portion of the readout sign 20 will appear aswell.

The devices of FIGS. 1 and 2 show a preferred format for the detectionof a single analyte or a plurality of analytes, respectively. Suchdevices can be prepared and used in the following manner.

Gold-KLH. Keyhole limpit hemocyanin ("KLH") and colloidal gold areobtained from a variety of commercial sources, and conjugated accordingto standard methods.

Gold-ovalbumin-drugs. Ovalbumin is obtained from a commercial source andcoupled to the desired analyte as well as to colloidal gold. Labeling ofthe carrier protein with colloidal gold is performed by standardmethods. Reaction conditions are monitored in order to ensure that thecolloidal gold does not interfere with the binding reaction of drughapten to anti-drug antibody. Similar chemical methods are used forpreparing conjugates for THC (marijuana), benzoylecgonine (cocaine),opiates (morphine, morphine glucuronide), amphetamine (amphetamine andmethamphetamine).

Nondiffusively bound reagents. Reagents can be immobilized to thebibulous material via any suitable technique as will be apparent tothose skilled in the art. Direct attachment methods include nondiffusiveadsorption, nondiffusive absorption, attachment to microparticles thatare themselves entrapped in the appropriate position, and covalentbinding, such as by use of cyanogen bromide, carbonyl diimidazole, orglutaraldehyde. "Nondiffusive", as used in this respect, means that thereagent is sufficiently stable in its position under the conditions ofthe assay.

Diffusively positioned reagents. Conventional methods are employed forimpregnating substrates such as paper with dry chemistry biomolecules.These methods are useful for: 1) optimizing substrate capacity; 2)optimizing the wettability of dried reagents; and 3) increasing thestability of dried reagents.

Indicator Strip. Conventional methods are employed for the preparationof a terminal indicator, for instance, by the use of a pH indicator thatwill change color when urine is present.

In one embodiment of the device, the diffusively positioned reagents areapplied to the competition zone in the appropriate concentrations suchthat a visibly perceptible signal is generated in the readout zone onlywhen the sample applied to the origin site contains analyte at or abovea predetermined concentration. When multiple analytes are being detectedfrom the same sample this predetermined concentration can be differentfor each analyte.

The operation of the device can be evaluated, for instance, by preparingspiked single drug samples in buffers, using varying concentrations ofeach drug. Limited cross-reactivity should be tested against otherabused substances as well as various common prescription drugs. Spikedmulti-drug samples are also tested, with particular emphasis on signalgeneration and possible signal interference. Signal generation can be"scored" visually against a standard color chart.

In one embodiment, normal human urine is spiked with varying doses ofthe four drugs of abuse. A total of five sources are used. Evaluationincludes comparison of: 1) visually scored signal generation; 2) timefor test completion; and 3) the presence of non-specific binding.

The method of the present invention comprises the steps of providing adevice of the type described above comprising a bibulous material in thefollowing manner, and:

(a) applying an aqueous sample to the bibulous material at a the originsite of one or more flow paths,

(b) allowing the sample to simultaneously flow through each flow pathand sequentially through a plurality of respective reagent zonesproviding the reagents necessary for performing a visual readout,competitive immunoassay for the presence of the respective analyte, thezones comprising, in order and in the direction of flow:

(i) a competition zone in fluid communication with the origin site andcomprising a detectable analyte-conjugate and binding partner for theanalyte, both being diffusively positioned in such a manner that anyfree analyte present in the sample is capable of binding to the bindingpartner in a competitive manner with the analyte conjugate;

(ii) a retention zone in latent fluid communication with the competitionzone and comprising an excess amount of a nondiffusively bound capturereagent capable of binding to free or bound binding partner in order toremove it from continued flow in the flow path; and

(iii) a readout zone in fluid communication with the retention zone andcomprising nondiffusively bound receptor capable of binding toanalyte-conjugate but not to unconjugated analyte in a detectablemanner;

(c) allowing the sample to flow through a terminal site downstream fromthe sequential reagent zones and comprising an indicator reagent forconfirming the completion of the respective assay,

(d) determining the presence of each analyte in the sample by detectingthe presence of the respective analyte-conjugate in each readout zone,and assessing the positive and negative controls in each flow path inorder to determine the proper performance of the respective assay.

What is claimed is:
 1. A device for performing a competitive immunoassayto provide a positive readout for the presence of a monoepitopic orpolyepitopic analyte in a sample, the device comprising a bibulousmaterial providing one or more flow paths, each flow path comprising:(a)a common origin site on the bibulous material for receiving a fluidsample, (b) a plurality of reagent zones emanating from the origin siteand providing the reagents necessary for performing a visual readout,competitive immunoassay for the presence of the respective analyte, thezones comprising, in order and in the direction of flow:(i) acompetition zone in fluid communication with the origin site andcomprising a detectable analyte-conjugate and binding partner for theanalyte, both being diffusively positioned in such a manner that freeanalyte present in the sample is capable of binding to the bindingpartner in a competitive manner with the analyte-conjugate; (ii) aretention zone in fluid communication with the competition zone andcomprising an excess amount of a nondiffusively bound capture reagentcapable of binding to free or bound binding partner in order to removeit from continued flow in the flow path; and (iii) a readout zone influid communication with the retention zone and comprisingnondiffusively bound receptor capable of binding in a detectable mannerto a conjugate of analyte and label but not capable of binding tounconjugated analyte.
 2. A device according to claim 1 furthercomprising one or more positive and/or negative procedural control zonesand reagents in fluid communication with one or more of the reagentzones.
 3. A device according to claim 2 wherein the positive controlzone comprises a terminal site downstream from the sequential reagentzones and comprising an indicator reagent for confirming the completionof the respective assay.
 4. A device according to claim 1 furthercomprising an inert holder comprising top and bottom portions forsupporting the bibulous material, the top portion providing an openingfor fluid sample access to the common origin site, as well as openingsfor viewing the respective readout and terminal indicator sites alongeach flow path.
 5. A device according to claim 1 wherein the analyte isselected from the group consisting of tetrahydrocannabinol, cocaine,opiates, and amphetamines.
 6. A method of performing a competitiveimmunoassay to provide a positive readout for the presence of amonoepitopic or polyepitopic analyte in a sample, the method comprisingthe steps of:(a) providing a device comprising a bibulous materialhaving an origin site and one or more flow paths each comprisingrespective reagent zone providing the reagents necessary for performinga visual readout, competitive immunoassay for the presence of therespective analyte, (b) and applying an aqueous sample to the originsite, (c) allowing the sample to simultaneously flow through each flowpath and sequentially through the respective reagent zone providing thereagents necessary for performing a visual readout, competitiveimmunoassay for the presence of the respective analyte, the zonescomprising, in order and in the direction of flow:(i) a competition zonein fluid communication with the origin site and comprising a detectableanalyte-conjugate and binding partner for the analyte, both beingdiffusively positioned in such a manner that any free analyte present inthe sample is capable of binding to the binding partner in a competitivemanner with the analyte conjugate; (ii) a retention zone in fluidcommunication with the competition zone and comprising an excess amountof a nondiffusively bound capture reagent capable of binding to free orbound binding partner in order to remove it from continued flow in theflow path; and (iii) a readout zone in fluid communication with theretention zone and comprising nondiffusively bound receptor capable ofbinding in a detectable manner to a conjugate of analyte and label butnot capable of binding to unconjugated analyte; (d) allowing the sampleto flow through a terminal site downstream from the sequential reagentzones and comprising an indicator reagent for confirming the completionof the respective assay, (e) determining the presence of the analyte inthe sample by detecting the presence of the respective analyte-conjugatein each readout zone, and assessing the completion of the assay in eachflow path in order to determine the proper performance of the respectiveassay.
 7. A device for simultaneously performing a plurality ofcompetitive immunoassays to provide a positive readout for the presenceof respective analytes in a sample, the device comprising a unitarybibulous material providing one or more flow paths, each flow pathcomprising:(a) a common origin site on the bibulous material forreceiving a fluid sample, (b) a plurality of respective reagent zonesdownstream from the origin, the reagent zones of each immunoassayproviding the reagents necessary for performing a visual readout,competitive immunoassay for the presence of the respective analyte, thezones comprising, in order and in the direction of flow:(i) acompetition zone comprising a detectable analyte-conjugate and bindingpartner for the analyte, both being diffusively positioned in such amanner that any free analyte present in the sample is capable of bindingto the binding partner in a competitive manner with the analyteconjugate; (ii) a retention zone comprising an excess amount of anondiffusively bound capture reagent capable of binding to free or boundbinding partner in order to remove it from continued flow in the flowpath; and (iii) a readout zone comprising nondiffusively bound receptorcapable of binding in a detectable manner to a conjugate of analyte andlabel but not capable of binding to unconjugated analyte.
 8. A deviceaccording to claim 7 further comprising one or more positive and/ornegative procedural control zones and reagents in fluid communicationwith one or more of the reagent zones.
 9. A device according to claim 8wherein the positive control zone comprises a terminal site downstreamfrom the sequential reagent zones and comprising an indicator reagentfor confirming the completion of the respective assay.
 10. A deviceaccording to claim 7 further comprising an inert holder comprising topand bottom portions for supporting the bibulous material, the topportion providing an opening for fluid sample access to the commonorigin site, as well as openings for viewing the respective readout andterminal indicator sites along each flow path.
 11. A device according toclaim 7 wherein the device provides a separate, discrete flow path foreach analyte, the flow paths being positioned to extend in a radialdirection from the common origin.
 12. A device according to claim 7wherein the assay is capable of differentially detecting the presence oftwo or more drugs in a single sample of urine or serum.
 13. A deviceaccording to claim 12 wherein the analytes are selected from the groupconsisting of tetrahydrocannabinol, cocaine, opiates, and amphetamines.14. A device according to claim 7 wherein a spacer moiety is used toconjugate the analyte to the label, and the nondiffusively boundreceptor is capable of binding to the spacer moiety.
 15. A deviceaccording to claim 14 wherein the label comprises a colloidal metalparticle.
 16. A method for simultaneously performing a plurality ofcompetitive immunoassays to detect the presence of respectivemonoepitopic or polyepitopic analytes in a sample, the method comprisingthe steps of providing a device comprising:(a) a common origin site onthe bibulous material for receiving a fluid sample, (b) a plurality ofrespective reagent zones downstream from the origin, the reagent zonesof each immunoassay providing the reagents necessary for performing avisual readout, competitive immunoassay for the presence of therespective analyte, the zones comprising, in order and in the directionof flow:(i) a competition zone comprising a detectable analyte-conjugateand binding partner for the analyte, both being diffusively positionedin such a manner that any free analyte present in the sample is capableof binding to the binding partner in a competitive manner with theanalyte conjugate; (ii) a retention zone comprising an excess amount ofa nondiffusively bound capture reagent capable of binding to free orbound binding partner in order to remove it from continued flow in theflow path; and (iii) a readout zone comprising nondiffusively boundreceptor capable of binding in a detectable manner to a conjugate ofanalyte and label but not capable of binding to unconjugatedanalyte,wherein the method comprises the steps of: (a) applying anaqueous sample to the unitary bibulous material at a common origin siteof one or more flow paths, (b) allowing the sample to simultaneouslyflow through each flow path and sequentially through a plurality ofrespective reagent zones providing the reagents necessary for performinga visual readout, competitive immunoassay for the presence of therespective analyte.
 17. A method according to claim 16 wherein themethod is capable of simultaneously performing a plurality ofimmunoassays to detect the presence of respective analytes in a sample,wherein the presence of increasing amounts of free analyte in a sampleleads to the binding of less analyte-conjugate to the binding partner.